Seed oil content has traditionally been modified by plant breeding. The use of recombinant DNA technology to alter seed oil composition can accelerate this process and in some cases alter seed oils in a way that cannot be accomplished by breeding alone. The oil composition of Brassica has been significantly altered by modifying the expression of a number of lipid metabolism genes. Such manipulations of seed oil composition have focused on altering the proportion of endogenous component fatty acids. For example, antisense repression of the .DELTA.12-desaturase gene in transgenic rapeseed has resulted in an increase in oleic acid of up to 83%. Topfer et al. 1995 Science 268:681-686.
There have been some successful attempts at modifying the composition of seed oil in transgenic plants by introducing new genes that allow the production of a fatty acid that the host plants were not previously capable of synthesizing. Van de Loo, et al. (1995 Proc. Natl. Acad. Sci USA 92:6743-6747) have been able to introduce a .DELTA.12-hydroxylase gene into transgenic tobacco, resulting in the introduction of a novel fatty acid, ricinoleic acid, into its seed oil. The reported accumulation was modest from plants carrying constructs in which transcription of the hydroxylase gene was under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Similarly, tobacco plants have been engineered to produce low levels of petroselinic acid by expression of an acyl-ACP desaturase from coriander (Cahoon et al. 1992 Proc. Natl. Acad. Sci USA 89:11184-11188).
The long chain fatty acids (C18 and larger), have significant economic value both as nutritionally and medically important foods and as industrial commodities (Ohlrogge, J. B. 1994 Plant Physiol. 104:821-826). Linoleic (18:2 .DELTA.9,12) and .alpha.-linolenic acid (18:3 .DELTA.9,12,15) are essential fatty acids found in many seed oils. The levels of these fatty-acids have been manipulated in oil seed crops through breeding and biotechnology (Ohlrogge, et al. 1991 Biochim. Biophys. Acta 1082:1-26; Topfer et al. 1995 Science 268:681-686). Additionally, the production of novel fatty acids in seed oils can be of considerable use in both human health and industrial applications.
Consumption of plant oils rich in .gamma.-linolenic acid (GLA) (18:3 .DELTA.6,9,12) is thought to alleviate hypercholesterolemia and other related clinical disorders which correlate with susceptibility to coronary heart disease (Brenner R. R. 1976 Adv. Exp. Med. Biol. 83:85-101). The therapeutic benefits of dietary GLA may result from its role as a precursor to prostaglandin synthesis (Weete, J. D. 1980 in Lipid Biochemistry of Fungi and Other Organisms, eds. Plenum Press, New York, pp. 59-62). Linoleic acid(18:2) (LA) is transformed into gamma linolenic acid (18:3) (GLA) by the enzyme .DELTA.6-desaturase.
Few seed oils contain GLA despite high contents of the precursor linoleic acid. This is due to the absence of .DELTA.6-desaturase activity in most plants. For example, only borage (Borago officinalis), evening primrose (Oenothera biennis), and currants (Ribes nigrum) produce appreciable amounts of linolenic acid. Of these three species, only Oenothera and borage are cultivated as a commercial source for GLA. It would be beneficial if agronomic seed oils could be engineered to produce GLA in significant quantities by introducing a heterologous .DELTA.6-desaturase gene. It would also be beneficial if other expression products associated with fatty acid synthesis and lipid metabolism could be produced in plants at high enough levels so that commercial production of a particular expression product becomes feasible.
As disclosed in U.S. Pat. No. 5,552,306, a cyanobacterial .DELTA..sup.6 -desaturase gene has been recently isolated. Expression of this cyanobacterial gene in transgenic tobacco resulted in significant but low level GLA accumulation. (Reddy et al. 1996 Nature Biotech. 14:639-642). Applicant's copending U.S. application Ser. No. 08,366,779, discloses a .DELTA.6-desaturase gene isolated from the plant Borago officinalis and its expression in tobacco under the control of the CaMV 35S promoter. Such expression resulted in significant but low level GLA and octadecatetraenoic acid (ODTA or OTA) accumulation in seeds. Thus, a need exists for a promoter which functions in plants and which consistently directs high level expression of lipid metabolism genes in transgenic plant seeds.
Sunflower embryos accumulate two major classes of storage proteins. These are the 11S globulins, soluble in 1M NaCl, and 2S albumins, soluble in water (Youle et al. 1981 Am J. Bot 68:44-48). The synthesis, processing and accumulation of 2 S albumin seed proteins have been studied intensively in Brassica napus (Crouch et al., 1983 J. Mol. Appl. Genet. 2:273-284; Ericson et al., 1986 J. Biol. Chem. 261:14576-14581), pea (Higgins et al., 1986 Plant Mol. Biol. 8:37-45), radish (Laroche-Raynal et al., 1986 Eur. J. Biochem. 157:321-327), castor bean (Lord J. M., 1985 Eur. J. Biochem 146:403-409) and Brazil nut (Sun et al., 1987 Eur. J. Biochem 162:477-483). A major conclusion of these studies is that the characteristic low molecular weight, disulfide-linked albumin polypeptides found in mature seeds result from the extensive processing of larger precursors synthesized during embryogenesis. Two additional characteristics that define the 2S albumin seed storage proteins are high amide content and high frequency of cysteine residues (Youle et al., 1981).
In sunflower, the 2S albumins represent more than 50% of the protein present in seeds (Youle et al., 1981) and consist of two or three closely related polypeptides with molecular weights of approximately 19 kDa (Cohen, E. A., 1986 "Analysis of sunflower 2S seed storage protein genes" MS thesis, Texas A&M University; Allen et al. 1987 Plant Mol Biol 5:165-173). The sunflower albumin is apparently maintained in a compact structure by intramolecular disulfide bonds resulting in a rapidly migrating species with an apparent molecular weight of 14 kDa when analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. When reduced, this species migrates as a 19 kDa polypeptide (Cohen, E. A., 1986). In contrast, most other 2S proteins are composed of large and small subunit polypeptides, derived from a single precursor, and linked by itnermoleclar disulfide bonds (Crouch et al. 1983 J. Mol. Appl. Genet. 2:273-284; Ericson et al. 1986 J. Biol. Chem. 261:14576-14581; Sun et al. 1987, Eur. J. Bioch. 162:477-483. )
Albumin polypeptides can be detected in sunflower embryos by 5 days post-fertilization (DPF), 2 days before helianthinins are detectable, and continue to accumulate through seed maturation. Sunflower albumin mRNAs, also first detected at 5 DPF, accumulate rapidly in sunflower embryos reaching maximum prevalence between 12 and 15 DPF. After this time albumin transcripts decrease in prevalence with kinetics similar to that observed for helianthinin mRNA (Allen et al. 1987). Functional sunflower albumin mRNAs are undetectable in dry seeds, germinated seedlings or leaves (Cohen 1986).
A number of albumin cDNAs and genomic clones have been isolated from different plant species including sunflower (Allen et al. 1987 Mol-Gen Genet. 210:211-218) and pea (Higgins et al. 1986 J. Biol. Chem 261:11124-11130). As in other classes of seed proteins such as Brassica napis (Crouch et al., 1983; Ericson et al., 1986), 2S albumin seed proteins are encoded by small gene families.
The present invention provides 5' regulatory sequences from a sunflower albumin gene which direct high level expression of lipid metabolism genes in transgenic plants. In accordance with the present invention, chimeric constructs comprising a sunflower albumin 5' regulatory region operably linked to coding sequence for a lipid metabolism gene such as a .DELTA.6-desaturase gene are provided. Transgenic plants comprising the subject chimeric constructs accumulate GLA to approximately 10% of C18 fatty acids. This is within the range of accumulation of GLA for Oenothera biennis, a primary commercial source for GLA.